Molecular Techniques and Methods

Isolation of Mitochondrial DNA
from Green Fruits

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In this procedure, mitochondria is separated by differential centrifugation from the bulk of nuclei, plastids, and cellular debris which differ in particle size or density. Mitochondria are then further separated from the remaining plastids and nuclear debris by gradient centrifugation. Homogenization media for mitochondrial isolation contain an osmoticum, EDTA, bovine serum albumin (BSA), 2-mercaptoethanol, and polyvinylpyrrolidone (PVP) which binds phenolics.

  • Yield of mtDNA; 5-10 ug/ 100 g Green Fruits





  • MATERIALS AND SOLUTIONS

    Fruit Grinding Buffer (1 liter)
    0.3 M mannitol --------------------------------- 54.7 g
    50 mM Tris-HCl (pH 8.0) ---------------------- 50 ml of 1 M Tris-HCl
    10 mM EGTA ---------------------------------- 20 ml of 0.5 M EGTA
    5 mM EDTA ----------------------------------- 10 ml of 0.5 M EDTA
    0.2% BSA -------------------------------------- 2 g
    1.0% PVP -------------------------------------- 10 g
    50 mM 2-mercaptoethanol ---------------------- 3.5 ml of 14.4 M 2-mercaptoethanol
    Deionized H2O to make a final volume of ------ 1 liter


    DNase Digestion Buffer (1 liter)
    0.3 M mannitol --------------------------------- 54.7 g
    50 mM Tris-HCl (pH 8.0) ---------------------- 50 ml of 1 M Tris-HCl
    10 mM EGTA ---------------------------------- 20 ml of 0.5 M EGTA
    5 mM EDTA ----------------------------------- 10 ml of 0.5 M EDTA
    0.2% BSA -------------------------------------- 2 g
    50 mM MgCl2 ---------------------------------- 50 ml of 1 M MgCl2
    Deionized H2O to make a final volume of ------ 1 liter


    DNase I (10 mg/ml)


    Gradient Buffer (1 liter)
    0.3 M Sucrose ------------------------------------ 102.7 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 50 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 1 g
    Deionized H2O to make a final volume of -------- 1 liter


    1.6 M Sucrose Step Gradient Buffer (100 ml)
    1.6 M Sucrose ------------------------------------ 54.8 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml



    1.2 M Sucrose Step Gradient Buffer (100 ml)
    1.2 M Sucrose ------------------------------------ 41.1 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml


    0.6 M Sucrose Step Gradient Buffer (100 ml)
    0.6 M Sucrose ------------------------------------ 20.6 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml


    ET buffer (100 ml)
    20 mM EDTA ------------------------------------- 4 ml of 0.5 M EDTA
    50 mM Tris-HCl (pH 8.0) ------------------------- 5 ml of 1 M Tris-HCl
    Deionized H2O ------------------------------------ 91 ml




    PROCEDURES

  • All steps should be performed in ice!

    Ioslation of Mitochondria from Green Fruits

    1. Collect young green fruits which are a reliable source of mtDNA.
  • Mitochondrial number may become smaller during ripening, since mitochondrial protein, expressed as a percentage of cytoplasmic protein, decreases during fruit maturations. In ripe fruit, compounds produced also damage mitochondria during isolation.

    2. Wash green fruits with tap water and chop into small pieces.

    3. Add 2 volumes of Fruit Grinding Buffer per volume of fruit.

    4. Grind 1,500 g of green fruits in a waring blender briefly.

    5. Filter the homogenate through two layers of cheesecloth and one layer of Miractoth.

    6. Adjust the pH to 7.5 with 5 M NaOH.

    7. Centrifuge the homogenate for 15 minutes at 1,500g and 4oC.

    8. Filter the supernatant through one layer of Miracloth.

    9. Harvest the crude mitochondria by centrifuging for 15 minutes at 13,000g and 4oC.

    10. Resuspend the pellet gently with a paint brush in 12 ml of DNase Digestion Buffer.

    11. Digest nuclear DNA with 120 ul DNase I (10 mg/ml) on ice for 45 minutes.

    12. End the DNase treatment by adding 4 ml 0.5 M EDTA.

    13. Dilute the preparation with 400 ml of Gradient Buffer.

    14. Collect the mitochondria by centrifuging for 15 minutes at 13,000g and 4oC.

    15. Resuspend mitochondrial pellet in 12 ml of Gradient Buffer.

    16. Layer 3-4 ml of mitochondrial suspension per sucrose step gradient consisting of 10 ml 1.6 M Sucrose Step Gradient Buffer, 10 ml 1.2 M Sucrose Step Gradient Buffer, and 10 ml 0.6 M Sucrose Step Gradient Buffer.

    17. Centrifuge for 1 hour at 25,000 rpm for purification from contaminating subcellular structures and residual DNase.

    18. After centrifugation, the bands at the interface will appear green because of thylakoid contamination. Collect the lower band.

    19. Dilute the mitochondrial fraction with 3 volumes of Gradient Buffer slowly over a 15-minute period to minimize disruption by osmotic shock.

    20. Harvest the mitochondria by centrifugation at 15,000g for 10 minutes at 4oC.

    21. Resuspend the mitochondrial pellet in 7 ml of ET buffer.

    22. Add 350 ul of 10% Sarkosyl to lyse the mitochondria.
  • Optional; add 1 ul of Proteinase K (20 mg/ml) and incubate for 15 minutes on ice to reduce clumps of debris.


    CsCl Gradients for mtDNA Purification

    23. To 7 ml of lysed mitochondria, add 8.05 g CsCl and 220 ul of ethidium bromide (10 mg/ml).

    24. Add ET buffer to make the total weight to 8.32 g. Dissolve the CsCl.

    25. Transfer the liquid to a Beckman Quick-Seal tube and heat-seal the tube.

    26. Centrifuge at 65,000 rpm for 8-10 hours at 20oC.

    27. Collect both linear and supercoiled DNA bands with a syringe.

    28. Extract the ethidium bromide three times with isopropanol equilibrated with CsCl-saturated TE buffer.

    29. Dialyze mtDNA against 4 liters of STE buffer for 1-2 hour.

    30. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of ethanol.

    31. Precipitate DNA at -20oC overnight.

    32. Collect DNA by centrifugation at 15,000 rpm at 4oC for 20 minutes.

    33. Resuspend mtDNA in 100-500 ul TE and store at -20oC or 4oC.




    NOTES

  • Deoxyribonuclease (DNase) is used to remove remaining DNA external to the mitochondria. The effectiveness of this DNase step requires penetration of the DNase into nonintact contaminating plastids and nuclear debris and sufficient mitochondrial integrity to prevent the enzyme from entering the organelles. Integrity of mitochondria is critical when utilizing a DNase step during purification. This treatment should be performed early in the isolation procedure while the highest proportion of mitochondria are intact.


  • In some mtDNA preparations, faint bands which comigrate with purified plastid DNA restriction fragments can be seen on ethidium bromide-stained gels of restriction enzyme-digested mtDNA preparations. Whether these represent plastid DNA contamination can be checked by hybridizing total plastid DNA (nick-translated) to a Southern blot.


  • If DNase treatment of sucrose gradient-purified fractions does not give adequate purification from plastid DNA, dyes which enhance separation of organelle DNAs can be incorporated into the CsCl gradients. Plastid DNA could be quantitatively separated from mtDNA on CsCl gradients containing diamidinophenylindol or bisbenzimide.





  • KIT INFORMATION




    REFERENCES

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  • DeBonte, LR, Matthews, BF (1984) Plant Mol. Biol. Rep. 2: 32.


  • Dixon, LK, Leaver, CJ (1982) Plant Mol. Biol. 1: 89.


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  • Hack, E, Leaver, CJ (1984) Curr. Genet. 8: 537.

  • Ku, HS, Pratt, HK, Spuff, AR, Harris, WM (1968) Plant Physiol. 43, 883.


  • Murray, MG, Thompson, WF (1980) Nucleic Acids Res. 8: 4321.


  • Palmer, D, Shields, CR (1984) Nature (London) 307: 437.

  • Powling, A (1981) Mol. Gen. Genet. 193: 82.


  • Qudtier, F, Vedel, F (1977) Nature (London) 268: 365.


  • Rigby, P, Dieckmann, M, Rhodes, C, Berg, P (1977) J. Mol. Biol. 113: 237.


  • Sparks, Jr, RB, Dale, RMK (1980) Mol. Gen. Genet. 190: 351.


  • Stern, DB, Palmer, JD (1984) PNAS 8: 1946.


  • Synenki, RM, Levings, CS, Shah, DM (1978) Plant Physiol. 61: 460.


  • Vedel, F, Mathieu, C (1982) Anal. Biochem. 127: 1.



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